Project Summary The alpha cells of the pancreatic islet are the primary source of glucagon in the body, which serves as the main counterregulatory hormone to beta cell-derived insulin. Glucagon mobilizes hepatic glucose production to increase blood glucose and protect from hypoglycemia, but in addition to reacting to glycemic status, alpha cell secretion is mediated by a multitude of other signals. Among the other alpha cell effectors are epinephrine, arginine vasopressin, oxytocin, and amino acids from outside the islet. The many inputs affecting alpha cells and their uneven distribution amongst other cell types in the islet has so far precluded researchers from generating a comprehensive understanding of how they function. This complexity is no more apparent than in diabetes, where, in conjunction with insulin impairment, glucagon dysfunction remains unaddressed despite nearly 50 years of research on the issue. A better mechanistic understanding of alpha cells is paramount to developing effective therapeutics to address the defects in diabetes. In the Huising Lab, I have pioneered a method of imaging alpha cell activity in intact islets in real time, and in doing so vastly increased the throughput of measurable alpha cell behavior. I have observed heterogeneous responsiveness of alpha cells to different stimuli previously assumed to uniformly activate all alpha cells within an islet. These observations have led to my hypothesis that alpha cells exist as a pool of functionally heterogeneous subpopulations that respond to different stimuli. The relative distribution of these subtypes is altered in the context of diabetes, contributing to the changes in glucagon secretion seen in disease. In this proposal, I aim to quantify heterogeneous responses by alpha cells in healthy and diabetic mouse and human islets and how this reflects on the total glucagon secretion. By successfully leveraging my high throughput live imaging of both calcium and cAMP in intact islets to define functional heterogeneity in alpha cell activation I will have established a new paradigm by which the field may need to think about not just alpha cells, but the islet cells in general. Successful demonstration of changes in alpha cell heterogeneity in the context of diabetes has the potential to inform on more effective methods by which to normalize glucagon release in disease.